SERUM PROTEINS IN CF

when 2 0 pI of sera from homozygotes o r heterozygotes were applied in the first step (IEFAG); however, they observed no protein from the same amount of control sera, except in hemolyzed samples, which had a protein with electrophoretic mobility identical to the protein from C F sera. O n the basis of the protein's cationic nature and relatively low molecular weight, Altland et nl. (2) also suggested that this protein is similar to, if not identical with, the ciliary dyskinesia factor. This communication relates our attempts to fractionate serum proteins by isoelectric focusing and electrophoresis and to detect a unique protein o r proteins in C F homozygous and hetcrozygous sera. The I E F A G method specified by Wilson et 01. (53) has been reproduced in our laboratory. The two-step IEFAG/ disc electrophoresis technique outlined by Altland et nl. (2) has also been employed in our analyses. Furthermore, we have incorporated several methodologic improvements into the isoelectric focusing technique. These improvements have enabled us to demonstrate significantly enhanced resolution and a greater degree of heterogeneity of serum y-globulins than other investigators have reported. T h e improved technique should increase the likelihood of detecting a CF-specific serum protein. MATERIALS A N D METHODS

C F patients were diagnosed on the basis of clinical history and an elevated chloride concentration in the sweat (20). Obligate heterozygotes were parents of these patients. Normal control subjects were clinically healthy volunteers with no known family history of CF. Several of the control subjects were orientals o r American blacks, in whom the C F g e n e frequency is low ( 2 4 , 2 9 , 4 9 , 56). Informed consent was obtained before subjects were admitted to the study. Venous blood was collected and allowed to clot in glass tubes at 4" for 2-4 hr. The blood was then centrifuged at 1,500 x g for 10 min at 4'. The serum was transferred to plastic tubes and either used fresh o r frozen in aliquots at -20' o r -70' for later analysis. The concentration of IgG was determined for some serum samples by single radial immunodiffusion (57) in order to standardize the volume of serum as specified by Wilson et nl. (53). Isoelectric focusing in thin layer polyacrylamide gel and the combined techniques of IEFAG/thin layer disc electrophoresis were performed in a plastic chamber with an aluminum cooling block (58) through which coolant was circulated at 4". The thin layer (1.5 mm thick) polyacrylamide gels (59) were formed between two glass plates (20 x 20 cm) by a common procedure (47, 50). Platinum ribbon electrodes (60) and Ampholine carrier ampholytes (61) were used in the I E F A G technique. Ten to 12 samples, including at least 3 of each genotype ( C F homozygote, C F heterozygote, normal control), were analyzed per gel to facilitate comparison of banding patterns. The p H gradients in I E F A G were measured at 8' with flat membrane microelcctrodes (62). IEFAG ACCORDING TO WILSON ET A L . (53)

The biophysical assay of Wilson el 01. was reproduced as described (53) with confirmation and elaboration of the methodology provided (51). The volume of serum samples analyzed by their technique was varied in order to have a constant amount of IgG (300 p g ) in each sample, as specified (53). We have applied their method to the analysis of sera from 16 C F patients, 13 obligate heterozygotes, and 14 normal control subjects. MODIFIED IEFAG (43)

A variety of methodologic approaches to analytic isoelectric focusing were investigated in order to maximize the resolution of serum proteins, particularly the y-globulins in the alkaline p H range. The details of our methods, a comparison with existing techniques for I E F A G , and the rationale for our approach to

1149

analytic isoelectric focusing will be presented elsewhere (43). In general, our attempts to detect a CF-specific protein in the isoelectric spectrum of serum proteins utilized three different p H gradient systems in IEFAG: ( 1 ) the standard broad range of p H 3.5-10, (2) a narrow range of pH 7-9, and (3) a narrow range of pH 8.5-10.5. In the broad range system ( p H 3.5-lo), the gel composition was as follows: T = 3 % and C = 9 % (63), 1.0% Ampholine carrier ampholytes ( p H 3.5-lo), 4.0 M urea, and 0.00005% riboflavin. Samples were pipetted onto 8-mm wide pads of chromatography paper (64) placed near the middle of the gel. Electrofocusing was performed in a constant power mode during the initial stages of the run, after starting with a power supply voltage of 1000 V for an 1 8 cm electrode distance; the final potential was 2000 V. The duration of the electrofocusing run was from 3.5-5.5 hr, depending o n the portion of the isoelectric spectrum to be emphasized (43). After electrofocusing, the gels were fixed in a hot acid-alcohol solution and the ampholytes removed before staining with Coomassie brilliant blue (43). The two alkaline range I E F A G systems had different gel compositions, but electrofocusing in these systems was based on the same principle. In the p H 7-9 system, the gel composition was as follows: T = 3 % and C = 9%, 1 .O% Ampholine carrier ampholytes (0.1% p H 3.5-10, 0.2% p H 5-7, 0.7% p H 7-9), 4 . 0 M urea, 15% (v/v) glycerol, and 0.00005% riboflavin. In the p H 8.5-10.5 system, the gel composition was as follows: T = 3% and C = 9 % , 1.2% Ampholine carrier ampholytes (0.2% p H 3.5-10, 0.4% p H 7-9, 0.6% p H 9-1 I ) , 0.20% oxalic acid dihydrate (added to neutralize the solution to enable photopolymerization), 4.0 M urea, 15% (v/v) glycerol, and 0.00005% riboflavin. These latter gels were prefocused to disperse the oxalate ions toward the anode before ample application. In both alkaline range systems electrofocusing was performed in two stages. The first stage, during which the p H gradient began to form and proteins began to migrate either cathodally o r anodally, was performed with the usual 1 8 cm electrode distance. For the second stage, the more acidic end of the gel was discarded and the anodic electrode was reapplied to the gel at a position in the gradient corresponding to p H 7.0 (about 14 cm from the cathodic electrode) in the p H 7-9 system o r p H 8.5 (about 10.5 cm from the cathodic electrode) in the p H 8.5-10.5 system. This rearrangement of the anodic electrode allowed a more even conductance course between the electrodes and thus a higher potential drop across the alkaline range of the gradient (43). The total duration of the electrofocusing run, with potentials reaching 2000 V , was 14 hr. The details of the methodology for focusing in the alkaline range will be presented elsewhere (43). In addition to the general protocols outlined above, we employed variations in our analyses which should increase the possibility of detecting any consistent difference o r differences among the genotypes. Gels of different pH gradients, with and without urea, were utilized. From 10-50 pI of whole serum were analyzed. Alternatively, a volume of serum was used which contained 3 0 0 p g IgG, as recommended by Wilson e t n l . (53). In some cases (as in the electrofocusing runs of shorter duration) the serum samples were pretreated with 4.0 M urea for 4-24 hr. We have applied o u r various methods to the analysis of sera from 22 C F patients, 2 3 obligate heterozygotes, and 21 normal control subjects. TWO-STEP IEFAGITHIN LAYER DISC ELECTROPHORESIS OFALTLAND ET A L. (2)

The essential aspects of the two-step, one-dimensional technique as described by Altland et nl. (2) were reproduced. The method of performing the first step (IEFAG to isolate IgG fractions) was varied. This preparative procedure was usually accomplished either in the manner specified by Altland et nl. (2) o r in a manner following our standard protocol as outlined above. Alternatively, the IEFAG step was performed as speci-

1150

THOMAS, MERRITT, AND HODES

fied by Wilson et al. (53), to simulate their conditions of sample treatment and fractionation. The second step (thin layer disc electrophoresis) as outlined by Altland et al. (2) was used in some analyses, but modifications were later introduced to improve the resolution. The major modifications included the following: a stacking gel with the potassium-acetate buffer at pH 6.5 (measured in the presence of 4.0 M urea at 22"); a separating gel of T = I S % , C = 5 % with the potassium-acetate buffer at pH 5.0 (measured in the presence of 4.0 M urea at 22O); rapid equilibration of the excised gel slab from IEFAG (pH 7.5-9.5) in a buffer solution identical to that of the stacking gel; a 5.5-6.5-hr electrophoretic run at a constant current of 40 mA; and staining of the separating gel by the method used in our IEFAG analyses (43). We originally analyzed 20 pl of sera by this two-step technique, as suggested by Altland er 01. (2). Since the degree of resolution in the first step (IEFAG) was not crucial, up to 200 p1 sera were analyzed in an effort to detect differences in protein bands present a t low concentration. We have applied the twostep IEFAGIdisc electrophoresis technique to the analysis of sera from 19 C F patients, 19 obligate heterozygotes, and 15 control subjects. RESULTS IEFAG ACCORDING TO WILSON ET A L . (53)

Based o n the quantitation of IgG in the sera, the ranges of sample volumes (containing 300 p g IgG) needed for analysis were as follows: 11-32 pI (mean, 20) for 19 CF, 14-33 p1 (mean, 25) for 20 heterozygote, and 13-44 p1 (mean, 27) for 21 normal control samples. These values fall within the ranges reported by Wilson et al. (53). The protein banding patterns and the pH gradients obtained by the method of Wilson et al. (53) appeared similar to those shown in their illustrations. Among the 16 CF, 1 3 heterozygous, and 14 normal control sera analyzed, there was no band unique to the C F genotypes. However, the resolution was poor compared to the resolution obtained with our methods (see below and Fig. 6). We conclude that the method of Wilson et al. (53), as reproduced in our laboratory, is not capable of distinguishing genotypes. MODIFIED IEFAG (43)

Generally 3 0 o r 40 pI sera were used in our IEFAG analyses. T h e concentration of IgG in some of the serum samples was determined for analysis by the method of Wilson et al. (53) o r by

Fig. 1. Isoelectric focusing in polyacrylamide gel patterns of serum proteins using the broad range gradient (pH 3.5-10). Only the alkaline end of the gel is shown. Sample volume was 30 pl. Serum samples are designated as: CF (cystic fibrosis), H (CF heterozygote), and N (normal control). Marker proteins are on right side: Mb (sperm whale myoglobin), Hb (human hemoglobin), C (horse heart cytochrome c). Urea concentration was 4.0 M. The measured pH gradient is shown to the

0 H CF

N

H CF N

H CFCF

N

Fig. 2. Isoelectrie focusing in polyacrylamide gel patterns of serum proteins usings a narrow range gradient (pH 7-9). Each sample contained 300 pg IgG. Serum samples and marker proteins (on left side) are designated as in Figure 1. Urea concentration was 4.0 M. The measured pH gradient is shown to the side.

our method, and these determinations (see above) indicated that a sufficient volume of serum was analyzed to enable the detection of the "CF factor protein (CFP)" (53). A n example of IEFAG using the broad range gradient (pH 3.5-10) is shown in Figure 1. Only the alkaline end of the gel is shown in this illustration, to emphasize the relevant section of the isoelectric spectrum. The banding pattern shows significant heterogeneity in this portion of the y-globulin fraction. This pattern also indicates the presence of numerous protein bands both near the region of pH 8.4-8.5 (at which sperm whale myoglobin focuses in 4.0 M urea) rcported to contain the C F factor protein (53) and in the more alkaline region. In fact, the most alkaline serum proteins in the isoelectric spectrum are located at a p H near 10 (as measured in 4.0 M urea). Thus, our method resolves numerous proteins in the far alkaline rcgion of the gradient which are not apparent in the illustrations of Wilson et 01. (53). Since this discrepancy, which is explainable in terms of the theoretic and practical aspects of IEFAG (see below), appeared consistently, we did not concentrate on detecting a difference near pH 8.4-8.5 but considered the entire alkaline portion of the spectrum in our analyses. Examples of IEFAG using the alkaline range gradients (pH 79 and pH 8.5-10.5) are illustrated in Figures 2 and 3 , respectively. These methods significantly increase the resolving power in the alkaline range and thus enable the demonstration of an even greater degree of heterogeneity within this portion of the isoelectric spcctrum of serum proteins. Also, using these methods of isoelectric focusing in two stages, larger sample volumes (50 p1 o r more) could be analyzed without engendering much distortion in the protein banding patterns, since the section of the gel containing the serum proteins in high concentration ( i . e . , albumin and a-2-macroglobulin) is discarded. These three methods and other variations in the IEFAG technique were applied to the analysis of 22 CF, 23 heterozygous, and 21 normal control sera. No consistent differences were apparent, despite a significant increase in the number of protein bands which were resolved. TWO-STEP IEFAGITHIN LAYER DISC ELECTROPHORESIS OF ALTLAND ET A L. (2)

The combined technique of IEFAGIthin layer disc electrophoresis enabled the separation of low molecular weight proteins from serum IgG. Thus, this approach should allow samples to be analyzed for the presence of small cationic proteins and should simplify the observation of the C F serum factor.

1151

SERUM PROTEINS IN CF A n example of results with the two-step, one-dimensional technique of Altland et nl. (2) is illustrated in Figure 4. This separating gel from the thin layer disc electrophoresis step demonstrates that at least one cationic protcin of relatively low molecular weight can be fractionatcd from all serum samplescontrol as well as C F sera (visibly unhemolyzed)-and that this protein has electrophoretic mobility identical to a larger amount of protcin observed in hemolyzed samplcs. The modifications which we introduced in the disc technique separate thesc proteins more efficiently, as illustrated in Figure 5 . Thc results indicate that several cationic protcins of low molecular weight can be fractionatcd from a portion of serum gamma globulins (approximately pl 7.5-9.5). Furthermore, benzidine staining indicates that some of thesc proteins contain heme. U p to 200 p1 . sera from 16 CF, 17 heterozygote, and 13 normal control samples were analyzcd by either the technique of Altlandet 01. (2) o r our modifications of this two-step technique. In addition, seven C F , five hcterozygous, and five normal control sera were analyzed by the two-step techniques employing the I E F A G mcthod of Wilson et nl. (53). No consistently unique cationic protein of low molecular weight was observed in samples from C F patients o r obligate heterozygotes.

c

-

H

CF

N

H

CF

N

H

CFCF

Fig. 5. Results of the twestepisoelectric focusing in polyacrylamide gcllthin layer disc electrophoresis, with our modifications. This illustration shows an enlargement of a portion of the thin layer separating gel (T = 15%). Sample volumc was 100 p1. Serum samples are designated as in Figurc 1. All samples were visibly unhemoly&d. Urea concentration was 4.0 M.

N -10.28

Fig. 3. ~soelcctricfocusing in polyacrylam~dcgel patterns of serum protcins using a narrow range gradient (pH 8.5-10.5). Each sample contained 300 pg IgG. Serum samples and marker proteins arc designated as in Figure 1. Urea concentration was 4.0 M. The measured pH gradient is shown to the side.

Fig. 6. Comparison of the four isoelectric focusing in polyacryli~mide gel techniques showing serum protein banding patterns in the alkaline range.A: comparison of patterns obtained by the mcthod of Wilson etal. (53) (IV) and by our method using the broad range gradient of pH 3.510 (1); B: comparison of patterns obtained by the method of Wilson et nl. (53) (IV) and by our method using the narrow range gradient of pH 7-9 (2); C: comparison of patterns obtained by the method of Wilson etnl. (53) (W) and by our method using the narrow range gradient of pH 8.5-10.5 (3). Samples shown are identical (from a cystic fibrosis hctcrozygote), and each contained 300 pg IgG. Urea concentration in each case was 4.0 M. Approximate pH gradients are shown to the side of each sample. The expansions of the isoclcctric spectra are indicated by the lines between each pair of banding patterns.

COMPARISON OF lEFAG TECHNIQUES

Fig. 4. Results of the two-step isoelectric focusing in polyacrylamide gellthin layer disc electrophoresis, as outlined by Altland et al. (2). Only the thin layer scparating the gel (T = 7.5%) is shown. Sample volume was 50 pl. Serum samples are designated as in Figure 1. The CF sample in the center was hemolyzed to facilitate a comparison with visibly unhemolyzed samples. Marker proteins are on right side. Urea concentrations was 4.0 M.

The improvement in the technique of analytic isoelcctric focusing should be obvious from inspection of Figure 6. This illustration shows a comparison of scrum protcin banding patterns in the alkaline range obtained by the four mcthods indicated. The method of Wilson el nl. (53), using a pH 5-10 gradient, results in a paucity of single, identifiable protein bands. Although our method using the broad range gradient of pH 3.510 shows significant improvement in the resolution and results in numerous identifiable bands, the methods which we have devised for focusing in the narrow gradients of pH 7-9 and p H 8.510.5 enhance the resolution even further. Consequently, the latter mcthods should be preferred for determining the banding pattern difference among individual sera with respect to yglobulins in the alkaline range of the isoelcctric spectrum. The improved resolution with the p H 7-9 o r p H 8.5-10.5 system over that with our typical pH 3.5-10 system is a consequence of the shallower p H gradicnt (a lowcr slope of the p H gradient,,

1152

THOMAS, MERRI'TT. A N D HODES

d(pH)/dX) (21) and the maintenance of a higher field strength (potential difference) exclusively across the alkaline range of the gradient (43). A s indicated previously, even our method using the p H 3.5-10 system resolves numerous protein bands in the far alkaline region which are not resolved by the method of Wilson et al. (53), using the narrower pH 5-10 gradient. In the technique of Wilson et a[. (53), focusing does not proceed to equilibrium, primarily because the polyacrylamide gels of T = 5% exhibit a significant molecular sieving effect on the large yglobulin molecules and also because an inadequate field strength is maintained across the alitaline region of the gel (43). DISCUSSION

This study involved electrophoretic analysis in an attempt to detect a C F serum factor. The "biophysical assay" of Wilson et al. (53) and the "non-biological technique" of Altland et ol. (2) were reproduced. In addition, an extensive investigation using our own techniques for analytic isoelectric focusing and disc electrophoresis was undertaken. Our approach, employing different electrophoretic techniques and varying the conditions of sample analysis, should increase the likelihood of detecting a protein o r proteins specific for the C F genotypes. Many of the serum samples in our study were analyzed by more than one technique. Thus, in many cases, the same sample was analyzed by the method of Wilson et al. (53), by the method of Altland et al. (2), and by our various methods. Furthermore, in some cases, different serum samples were obtained from the same individual in an attempt to determine any intraindividual variation. Despite the many variations in our approach, no consistently unique protein was observed in C F o r heterozygous sera. The "standardized biophysical assay" of Wilson et al. (53), as reproduced in our laboratory, did not enable the detection of a C F factor protein near pH 8.4-8.5. However, at the level of resolution obtained with this method, nuances in technique (e.g., the use of different batches of Ampholine chemicals) could conceivably obscure the band which may represent a protein unique to C F and heterozygous sera. Consequently, the IEFAG technique was improved to an extent capable of demonstrating striking heterogeneity in serum proteins (43). Despite the significant increase in the number of bands resolved by our methods, neither a difference at pH 8.4-8.5 nor other differences throughout the alkaline pH range could be detected consistently in the C F and heterozygous sera. However, since some unknown factor in the technique of Wilson et al. (53) might result in the "production" of the C F protein factor at pH 8.4-8.5, our investigations have included the use of thcir IEFAG method in the two-step IEFAG/disc electrophoresis technique. This combination of techniques also did not result in the detection of consistent differences. Thus, our efforts indicate, contrary to the implication by Wilson et al. (53), that a low molecular weight, cationic C F serum protein is not readily demonstrable. Furthcrmore, our IEFAG analyses did not support the usefulness of the "B, C , and D bands" reported by Wilson et al. (53) in distinguishing between C F and heterozygous sera. In view of the complexity of the banding pattern in this region of the spectrum (pH 7.5-8.5), a real difference relative to one of only three bands would appear to be unlikely. The two-step method of Altland et al. (2) might be invaluable if the C F serum factor were a low molecular weight, cationic protein. As indicated previously, the application of this technique would simplify the detection of such a protein band if the resolution at the IEFAG step were inadequate. Our investigations using the "non-biological technique" of Altland et a[. (2) indicated that at least one small, cationic protein could be fractionated from all serum samples. Improvements in the method of disc electrophoresis resulted in the observation of numerous bands from some samples and of differences among the samples, but no protein band unique to the C F genotypes was observed. Furthermore, the major cationic protein(s) fractionated from some sera in the second step appeared to correlate with the

presence of heme in the band(s), although these serum samples were visibly unhemolyzed. In their report, Altland et al. (2) stated that different serum samples from the same "factorpositive individual" showed "quantitative variation from zero to intensive even in 10 pI samples," with respect to the "CF factor band" which they obscrved. Some intraindividual variation was also noted in some of our samples, but, more importantly, the relevant band was intense only in those samples which were slightly hemolyzed. (Some liberation of hemoglobin from erythrocytes may be unavoidable when withdrawing and processing blood.) In view of the great quantitative variation noted by Altland et al. (2) and their application of the assay to only a few serum samples, their demonstration of a difference between C F and control sera may have resulted from a fortuitous difference in serum hemoglobin rather than from the actual observation of a C F serum factor. Recently, a preliminary report of this investigation appeared (42), together with a response by Wilson et nl. (55). In addition, Smith et RI. (39) reported the inability to reproduce the results of Wilson et ol. (53, 54) and Altland et ol. (2). Although we have not seen their response (52) to the latter report (39) at the time of this writing, Wilson et ol. (55) related that they had suggested (52) several possible reasons for the conflicting results we initially reported (41), and those of Smithet a[. (39). Also, Wilson et al. (55) stated that additional details were provided (52) concerning their methodology and the reagents employed. Presumably, their additional details will complete a description of the incomplete methodology presented earlier (53, 54). In their response (55) to our preliminary note (42), Wilson et al. (55) discussed several points concerning their method which allegedly presented problems for us and which may present problems for other investigators attempting to use analytic isoelectric focusing to detect "cystic fibrosis protein (CFP)." Neither space nor discretion will pcrmit a complete response to all of the remarks made by Wilson et al. (55) concerning our attempts to reproduce thcir method. However, it is apparent that our procedures as reported here followed thcir guidelines specified originally (53) and also most of the new specifications (55) for their erstwhile "standardized" assay. Thus, their procedures (53) of sample collection and quantitation of IgG have bcen followed. Concerning the localization of "CFP" on the gel, our criteria have been more flexible than their requirement for finding a specific difference at a certain pH or a certain distance from the anode. As emphasized in the present report, we did not concentrate on detecting a difference near pH 8.4-8.5, but considered the entire alkaline portion of the spectrum in our analyses. In fact, this approach was necessary in view of the differences in pH measurements and corresponding band locations obtained with our improved techniques (see Figure 6). Although we are aware of potential problems in the precise measurement of pH gradients in analytic isoelectric focusing, we are also confident that the measurcments obtained from our modified methods reflect accurate values for the pI's of the relevant proteins. We base this conclusion o n the pI's of a specific marker protein (sperm whale myoglobin) and-the most cationic serum proteins (those with the highest PI'S). Several investigations on isoelect~icfocusing of sperm whale myoglobin have demonstrated a pI of 8.1 + 0.1 for the major component of this protein in the ferric state (25, 28, 31-35). We also have obtained this value for our preparation of sperm whale myoglobin when it was focused in the absence of urea. It has been realized for some time that urea increases the pK values of dissociable groups and the measured pH of aqueous solutions, presumably by reducing the activity of hydrogen ions (12), and the fact that urea also increases the apparent p H of carrier ampholyte solutions and thus the measured pI in isoelectric focusing has been discussed by several investigators ( 2 3 , 3 5 , 4 0 , 44, 45). Although the degree of elevation of pH will depend on the urea concentration and the buffering system, our studies and the reports in the literature (23, 3 5 , 4 0 , 4 4 , 4 5 ) indicate that the measured pI of a carrier ampholyte in 4.0 M urea would be

SERUM PRO

*

increased by 0.3 0.1 p H units. Thus the pI of the major ogcncity in CF. In an attempt to determine the reasons for component of sperm whale myoglobin (ferric) measured in the diffcrences in results between the laboratories, we have commupresence of 4.0 M urea would be 8.4 + 0.2. As illustrated in nicated with both Wilson and Altland (1, 51). Altland (1) has Figures 1 , 2 , and 3 of this rcport, our measured pI for this indicated to us that his technique will require further modificaprotein, using three different pH gradients, agrees well with the tion before a C F serum factor can be rcproducibly demonexpected valucs based on reports in the literature (25, 28, 31- strated. A collaborative study with the Wilson group would be 35). Concerning the most cationic proteins in serum, the true particularly valuable in resolving the differences. O u r efforts to pI's of these protcins havc not bcen realized, primarily because establish such a study have not been successful to date. In the of the heretofore persistent problems in alkaline range isoelec- future, cooperation between the involved laboratories may be tric focusing in polyacrylamide gels (43,48). We have bcen able necessary for the proper evaluation of thcse techniques with to solve many of thcse problems, thus accounting for our unprcc- respect to CF. / ' ' edented degree of resolution of serum proteins with high pI's. A s /' suggested earlier in this report and detailed elsewhere (43), our CONCLUSION approach to isoelcctric focusing in the alkaline range is based on A considerable amount of indirect evidence indicates that the theoretical aspccts of resolving power in natural (ampholytc) p H gradients, the physical properties of commercial ampholytes fluids from C F patients and obligate heterozygotcs contain a in an electric field, and the physicochcmical properties of scrum factor o r factors which may be relevant to the pathophysiology of protcins which focus in the alkaline range. The exploitation, of CF. Much of the evidence indicates that the factor is a low these aspects is requisite for the near equilibrium focusing of molecular weight, cationic protein which in scrum is associated cationic scrum proteins which is achieved in our system and with IgG. Detection of such a protcin factor by electrophoretic analysis should be possible, and two studies reported in the which has not been accomplishcd by other investigators to d:te. Howcver, several published refercnccs to isoelectric focusing of literature, each employing a different electrophoretic technique, have claimed identification of a serum protein unique to C F scrum proteins in sucrose dcnsity gradients have provided &,idcnce that some major scrum protcins focus at p H 9.5 o r abovc genotypes. Our study reproduced the tcchniqucs of Wilson et (11. (53) in the absence of urea (6, 22, 36, 3 7 , 4 6 , 50). T h e measured p i , of these proteins in the presence of 4.0 M urea would be 9.8 o r \ (isoelectric focusing in polyacrylamide gel) and Altland et n/. (2) above. Thus our illustrations showing bands at pH 9.8 o r higher (IEFAG/thin layer disc electrophoresis) in an attempt to detect are cntircly credible, contrary to the implications of Wilson et (11. a C F factor in serum. In addition, several modifications were incorporated into thcse techniques in order to enhance the (55). In addition, Wilson et (11. (55) stated that a major problem resolution, and variations in the conditions of sample analysis might have been our failure to use the unique apparatus cm- were cmploycd in order to increase the likelihood of detecting a ployed by Awdeh et al. (3). Howcver, the usc o r importance of protcin or proteins specific for the C F genotypes. Our modified this apparatus was neither stated nor emphasized in the original I E F A G method enabled the demonstration of striking hcterogereports by Wilson et al. (53, 54), and they arc now employing neity in serum y-globulins, but no consistent differences in band(55) a diffcrent apparatus, the LKB Multiphor. Thus it would ing patterns between C F o r heterozygous sera and control sera appear to be unlikely that our use of the Brinkman clectrofocus- have bccn apparent. Our modified IEFAG/thin layer disc elecing apparatus prccludcs the detection of any "cystic fibrosis trophoresis system indicated that several cationic, low molecular can be fractionated from all serum samples, but factor." With rcspcct to thcir rcsults using the LKB Multiphor weight apparatus presentcd in thc rcsponsc of Wilson et al. (55) to our no consistently unique protcin has been obscrved in C F o r preliminary rcport (42). it should be noted that they havc also hcterozygous sera. Although several possibilities may account altered thcir "standardized biophysical assay" by employing a for the discrepancy bctween our results and those rcportcd in the different p H gradient and a diffcrent voltage scqucnce. Thus, literature, our present conclusion is that the C F scrum factor(s) these aspects of thcir "standardized" assay may also not be cannot be rcrtdily demonstrated by the electrophoretic techessential for detecting "CFP," contrary to their communication niques described o r our improvements thereof. to us (51) o r the implication in thcir rcsponse (55). Lastly, it appears that Wilson et (11. (55) havc bccn able to improve thcir REFERENCES A N D NOTES patterns shown carlier (53) by modifying thcir mcthod. Thcir 1. Altland, K.: Personal communication. banding patterns have now improved sufficiently to enable a 2 . Altland. K.. Schmidt. S . R . , Kaiser, G . , and Knoche, W.: Demonstration of a comparison with ours presented carlier (42) and in the present factor in the serum of homozygotes and heterozygotes for cystic fibrosis by a communication. A major hallmark of the alkaline isoelectric non-biological technique. Humangenetik, 28: 207 (1975). spectruni of scrum protcins is the blank space at about pH 9.0 in 3 . Awdeh. 2 . L.. Williamson, A . R . , and Askonas, B . A , : lsoelectric focusing in polyacrylamide gel and its application to immunoglobulins. Nature,219: 6 6 our illustrations (Figures 1, 2 , and 3). This space is also apparent (1 968). in the illustration in the response of Wilso'n et 01. (55). but 4 . Barnett. D . R . . Kurosky, A , . Bowman, B . H . , and Barranco. S . C . : Loss of appears at about p H 7.5 in their publication (primarily because the ciliary inhibitory effect of the cystic fibrosis factor following proteolytic focusing has not approached equilibrium). However. these areas digestion and heat dcnaturation. Tex. Rep. Biol. Mcd.. 31: 697 (1973). 5 . Barnett, K. R., Kurosky, A . , Bowman, B . H . , Hutchison, H . T . . Schmoyer, are identical with rcspcct to the protein banding patterns, and I.. and Carson, S . D.: Cystic fibrosis: Molecular weight estimation of the some appreciation for this may he obtained by inspection of ciliary inhibitor. Tex. Rep. Biol. Med.. 31: 703 (1973). Figure 6 in this rcport, in which we compare our three modified 6 . Barnett. K. R., Schanfield, M. S . , McCombs. M. L . . and Bowman. B . H.: methods to our reproduction of the first "standardized assay" lsoelectric focusing and IgG allotyping of the serum factor containing the cystic fibrosis ciliary inhibitor. Tex. Rep. Biol. Med., 31: 7 0 9 (1973). presentcd by Wilson et (11. (53). The number of countable bands 7 . Beratis, N . G . , Conover, J . H . , Conod. E. J . , Bonforte, R. J . . and Hirschcathodal to this space will attest to the relative levcls of cxperihorn. K.: Studies on ciliary dyskinesia factor in cystic fibrosis. 111. Skin ence and expertise which Wilson ef a/. and we have achieved in fibroblasts and cultured amniotic fluid cells. Pediat. Res. 7: 9 5 8 (1973). analytic isoelectric focusing. 8 . Bowman, B . H.: Introduction: Progress in research toward identifying the basic defect in cystic fibrosis. Tex. Rep. Biol. Med. 31: 611 (1973). Clcarly, the results of our extensive investigation d o not con9. Bowman, B . H . , Hirschhorn, K.. and Bearn, A . G . : Bioassays of cystic firm the findings of Wilson et al. (53) o r Altland et 01. ( 2 ) . If a fibrosis factor. Lancet. i: 4 0 4 (1974). protein factor can be readily demonstratcd in C F scra by clcctro- 10. Bowman. B . H . , Lankford, B . J . . Fuller, G . M., Carson, S . D., Kurosky, A . . phoretic analysis, several possibilities may account for our findand Barnett, D . R . : Cystic fibrosis: The ciliary inhibitor is a small polypep tide associated with immunoglobulin G . Biochem. Biophys. Res. Commun., ings: diffcrcnces in technique and/or the handling of scra, gross 64: 1310 (1975). variation in the concentration of any C F factor(s), the prcscnce 1 I . Bowman. B . H . , and Mangos, J . A , : Current concepts in genetics: Cystic of the same (but physiologically inactive) factor(s) in control sera fibrosis. N . Engl. J . Med. 294: 937 (1976). at a generally lower but variable concentration, o r gcnetic heter- 12. Bull, H . B . . Breese, K . . Ferguson, G. L., and Swenson. C . A , : The pH o f urea

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solutions. Arch. Biochem. Biophys. 104: 297 (1964). 13. Committee for a Study for Evaluation of Testing for Cystic Fibrosis: Report of the Committee for a Study for Evaluation of Testing for Cystic Fibrosis. J. Pediat. 88: 71 1 (1976). 14. Conneally, P. M., Merritt. A. D.. and Yu, P.: Cystic fibrosis: Population genetics. Tex. Rep. Biol. Med. 31: 639 (1973). 15. Conover, J. H., Beratis, N. G.. Conod. E. J., Ainbender, E., and Hirschhorn, K.: Studies on ciliary dyskinesia factor in cystic fibrosis. 11. Short term leukocyte cultures and long term lymphoid lines. Pediat. Res. 7: 224 (1973). 16. Crow, J. F.: Problems of ascertainment in the analysis of family data. In: J. V. Neel, M. W. Shaw, and W. J. Schull: United States Public Health Service Publication, No. 1163, p. 23 (1965). 17. Danes, B. S.. Litwin. S. D.. Hutteroth, T. H.. Cleve, H., Bearn, A. G.: Characterization of cystic fibrosis factor and its interaction with human immunoglobulin. J. Exp. Med. 137: 1538 (1973). 18. Danks, D. M., Allan, J., and Anderson, C. M.: A genetic study of fibrocystic disease of the pancreas. Ann. Hum. Genet. 28: 323 (1965). 19. di Sant'Agnese. P. A., and Davis. P. B: Research in cystic fibrosis. N. Engl. J. Med., 295: 481, 534. 597 (1976). 20. Gibson, L. E., and Cooke. R. E.: A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics, 23: 545 (1959). 21. Haglund, H.: Isoelectric focusing in pH gradients-A technique for fractionation and characterization of ampholytes. Methods Biochem. Anal. 19: l (1971)., 22. Howard. A., and Virella. G.: The separation of pooled human IgG into fractions by isoelectric focusing, and their electrophoretic and immunological properties. In: H. Peeters: Protides of the Biological Fluids, Vol. 17, p. 449 (Pergamon Press, New York. 1969). 23. Josephsen, R. V.. Maheswaran, S. K., Mori, C. V., Jenness, R., and Lindorfer, R. K.: Effect of urea on pI's of ampholytes and casein in isoelectric focusing. Anal. Biochem. 40: 476 (1971). 24. Kulczcki, L. L.. Guin, G . H., and Mann, N.: Cystic fibrosis in Negro children: Results of a search. Clin. Pediat., 3: 692 (1964). 25. LaGow. J., and Parkhurst, L. J.: Kinetics of carbon monoxide and oxygen binding for eight electrophoretic components of sperm-whale myoglobin. Biochem. 11: 4520 (1972). 26. Lobeck. C. C.: Cystic fibrosis. In: J. B. Stanbury. F. B. Wyngaarden, and D. S. Frederickson: The Metabolic Basis of Inherited Disease, Ed. 3 (McGrawHill, New York, 1972). 27. McCombs, M. L.: Research in cystic fibrosis: A review. Tex. Rep. Biol. Med. 31: 615 (1973). 28. Nakhleh. E. T.. Abu Samra, S.. and Awdeh, 2. L.: lsoelectric focusing of phenanthroline iron complexes and their possible use as pH markers. Anal. Biochem. 49: 218 (1972). 29. Oppenheimer. E. H., and Esterly. J. R.: Cystic fibrosis in non-caucasian oatients. Pediatrics. 42: 547 (1968)., 30. P ~ I IM.~ J.. ~ ,and A. G.: Cystic fibrosis: Current concepts. J. Med. Genet.. 11: 249 (1974). . , 31. Radola, B. J.: Analytical and preparative isoelectric focusing of proteins in Sephadex and Bio-Gel layers. In: H. Peeters: Protides of the Biological Ruids, Vol. 18, p. 487 (Pergamon Press, New York, 1970). 32. Radola, B. J.: Isoelectric focusing in layers of granulated gels. I. Thin-layer isoelectric focusing of proteins. Biochim. Biophys. Acta 295: 412 (1973). 33. Radola, B. J.: Analytical and preparative isoelectric focusing in gel-stabilized layers. Ann. N. Y. Acad. Sci. 209: 127 (1973). 34. Radola, B. J.: lsoelectric focusing in layers of granulated gels. 11. Preparative isoelectric focusing. Biochim. Biophys. Acta 386: 181 (1974). 35. Salaman, M. R., and Williamson, A. R.: lsoelectric focusing of proteins in the native and denatured states: anomalous behavior of plasma albumin. Biochem. J. 122: 93 (1971). 36. Schmoyer, I. R.. Brooks, S. P., and Fischer, J. F.: Isolation andcharacterization of a ciliary dyskinetic factor from cystic fibrosis heterozygous serum. Life Sci. 11: 1037 (1972). 37. Schmoyer, I. R., Fischer. J. F.. and Brooks, S. P.: Fractionation of oyster cilia inhibitor from cystic fibrosis heterozygote serum. Biochem. Biophys. Res. Commun. 46: 1923 (1972). 38. Shwachman, H.: Changing concepts in cystic fibrosis. Hosp. Prac. 9: 143 11974). ,39. Smith, Q. T., Hamilton, M. J., and Shapiro, B. L.: Letter to the Editor. Pediat. Res., 10: 999 (1976). 40. Smyth, C. J.. and Arbuthnott. J. P.: Prowrties of Closrridium oerh.inees (ivelchii) Type-A *toxin (phospholipase'~)purified by electrofocusing. J. Gen. Microbiol.. 7: 41 (1974).

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41. Thomas, J. M.. Merritt, A. D., and Hodes, M. E.: Electrophoreticanalysisof serum proteins in cystic fibrosis. In: Cystic Fibrosis Club Abstracts, Seventeenth Annual Meeting. p. 19 (Cystic Fibrosis Foundation, Atlanta, 1976). 42. Thomas. J. M.. Merritt. A. D.. and Hodes. M. E.: Letter to the Editor: Electrophoretic analysis of serum proteins in cystic fibrosis. Pediat. Res., 11: 138 (1977). 43. Thomas, J. M., and Hodes, M. E.: In preparation. 44. Ui. N.: Isoelectric ~ o i n tand s conformation of rotei ins. I. Effect of urea on the behavior of some proteins in isoelectric focising. Biochem. Biophys. Acta, 229: 567 (1971). 45. Ui, N.: Conformational studies on proteins by isoelectric focusing. Ann. N. Y. Acad. Sci. 209: 198 (1973). 46. Valmet. E.: Demonstration of the microheterogeneity of human serum gamma-globulin by isoelectric focusing. Sci. Tools 15: 8 (1968). 47. Vesterberg. 0.: Isoelectric focusing of proteins in polyacrylamide gels. Biochim. Biophys. Acta 257: 11 (1972). 48. Vesterberg. 0.: Some aspects of isoelectric focusing in polyacrylamide gel. In: J. P. Arbuthnott and J. A. Beeley: Isoelectric Focusing (Butterworths. London, 1975). 49. Wang, C. I., Sumi. W. T., Stanton. R., Kwok, S., and Yamazaki, J. N.: Cystic fibrosis in an Oriental child. N. Engl. J. Med., 279: 1216 (1968). 50. Williamson, A. R.: lsoelectric focusing of immunoglobulins. In: D. M. Weir: Handbook of Experimental Immunology. Ed. 2 (Blackwell Scientific Publishers, New York. 1973). 51. Wilson, G. B.: Personal communication. 52. Wilson, G . B., Amaud. P., MOnsher. M. T., and Fundcnbcrg, H.: Letter to to the Editor: Detection of cystic fibrosis protein by electrofocusing. Pediat. Res.. 10: 1001 (1976). 53. Wilson, G . B.. Fudenberg, H. H., and Jahn, T. L.: Studies on cystic fibrosis using isoelectric focusing. I. An assay for detection of cystic fibrosis homozygotes and heterozygote carriers from serum. Pediat. Res., 9: 635 (1975). 54. Wilson, G . B., Jahn, T. L., and Fonseca. J. R.: Demonstration of serum protein differences in cystic fibrosis by isoelectric focusing in thin-layer polyacrylamide gels. Clin. Chim. Acta. 49: 79 (1973). 55. Wilson. G. B., Monsher, M. T.. and Fudenberg. H.: Letter to the Editor: Additional notes on the use of analytical isoelectric focusing for the detection of cystic fibrosis protein in serum. Pediat. Res., 11: 139 (1977). 56. Wright. S. W., and Morton, N. E.: Genetic studieson cystic fibrosis in Hawaii. Amer. J. Hum. Genet. 20: 157 (1968). 57. Immunoglobulin G (IgG) Test Kit for the Quantitation of Human IgG by the Technique of Single Radial Immunodiffusion. Meloy Laboratories. Inc., Springfield, Virginia. 58. Desagamrinkman TLE Double Chamber. Brinkman Instruments, Westbury, New York. 59. Acrylamide and N,N'-methylenebisacrylamide were either electrophoresis grade or a standard grade which was recrystallized according to Loening, U. E.: The fractionation of high molecular-weight ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J.. 102: 251 (1967). Eastman Organic Chemicals, Rochester. N. Y. 60. DesagaIBrinkman platinum ribbon electrodes. Brinkman Instruments, Westbury, New York. 61. LKB Ampholine carrier ampholytes. LKB Produkter. A. B. Stockholm, Brooma, Sweden. 62. DesagaIBrinkman pH measuring accessories. Brinkman Instruments. Westbury. N. Y. 63. % T = grams of acrylamide + grams of bisacrylamide per dl solution; % C = 100 X grams of bisacrylamide per dl per %T. Nomenclature according to Hjerten, S.: "Molecular sieve" chromatography on polyacrylamide gels, prepared according to a simplified method. Arch. Biochem. Biophys. Suppl.. 1: 147 (1962). 64. S&S No. 470 A-C "ash-low" filter paper. Schleicher & Schuell. Inc.. Keene. N. H. 65. The assistance of Debbie Tabakin and Alice Pigg in collecting the specimens is aratefullv acknowledeed. 66. John-M. ~ h o m a swas supported by National Institutes of Health Training Grant P H s GM 1056. 67. This is Publication no. 76-25 from the Department of Medical Genetics and was supported in part by the Indiana University Human Genetics Center, P H s GM 21054. 68. Requests for reprints should be addressed to: M. E. Hodes, M.D., Department of Medical Genetics, Indiana University School of Medicine, 1100 West Michigan St.. Riley Research 241. Indianapolis. Indiana 46202 (USA). 69. Received for publication December 2, 1977. 70. Accepted for publication February 23, 1977. Printed in U.S.A.